Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which has been previously assumed to be achieved through the activation of phospholipase C-gamma-2 (PLC- gamma-2) via tyrosine phosphorylation. Genetic studies revealed that the tyrosine phosphorylation level of PLC-gamma-2 is not well correlated with its enzymatic activity, suggesting that other factors are also involved in mediating hydrogen peroxide-induced activation of PLC-gamma-2. Here, we provide pharmacological and genetic evidence showing that phosphatidylinositol 3-kinase (PI3K) partially (~40 percent) mediated hydrogen peroxide-stimulated PLC-gamma-2 activity through a mechanism independent of its tyrosine phosphorylation level. Wortmannin, a specific inhibitor of PI3K, partially inhibited hydrogen peroxide-induced calcium release and IP3 production, but failed to alter tyrosine phosphorylation of PLC-gamma-2. Overexpression of Brutons tyrosine kinase (Btk), which was activated by hydrogen peroxide, almost completely overcame the inhibitory effect of Wortmannin on calcium release. Btk formed a stable complex with several tyrosine-phosphorylated proteins only in Btk overexpressing cells upon hydrogen peroxide stimulation. Among them, PLC-gamma-2 was physically associated with Btk which was responsible for the enhanced tyrosine phosphorylation of PLC-gamma-2. Together, our findings provide clear evidence for a better understanding of the molecular mechanisms by which hydrogen peroxide activates PLC-gamma-2. - Bruton's tyrosine kinase, calcium release, inositol trisphosphate, phosphatidylinositol 3-kinase, phospholipase C-gamma-2